Gap junctions formed by connexins are known to be essential for maintaining metabolic homeostasis of eye lens. We as well as others have recently revealed a novel feature of the lens connexin, which was found to be functionally involved in promoting epithelial-fiber differentiation and lens development. Our long-range goal is to understand the regulatory mechanisms and functional significance of gap junctions and their component, connexins in lens development and homeostasis. The objective of this application is to understand the mechanistic roles of Cx45.6 in cell differentiation, and in lens growth and development. The central hypothesis is that Cx45.6, unlike two other types of lens connexins, is functionally involved in the differentiation process and the formation of mature lens fibers. This hypothesis has been formulated on the basis of our significant preliminary findings, which demonstrate that Cx45.6 expression enhances lens epithelial-fiber differentiation and Cx45.6 is posttranslationally regulated by caspase-3 that is actively involved in terminal lens differentiation. Three specific aims will be pursued: 1). Determine the functional involvement of Cx45.6 expression in epithelial-fiber differentiation and lens development; 2). Identify functional domain(s) of the Cx45.6 molecule important for the stimulatory effect on lens differentiation and characterize the interactions between Cx45.6 and MIP, and other potential factor(s), and 3). Determine the functional significance of caspase-3-mediated cleavage of Cx45.6. One of the innovative aspects is that this proposal aims to dissect the mechanisms of an unconventional function of lens connexin. Moreover, the research program will be accomplished using our established avian retroviral approaches as the principal tool for experimental manipulations of primary chick lens cultures as well as in vivo chick lenses. It is our expectation that our experimental findings will provide the molecular basis for understanding the functions and regulation of Cx45.6 in differentiating lens fibers and have a major impact on defining how gap junctions are involved in lens growth and development. The outcomes of our research will be significant because the new knowledge discovered will contribute to a broader understanding of gap junctions and connexins in cell differentiation and tissue development. Furthermore, this research activity should make a contribution to the therapeutic strategies for the treatment of eye diseases such as cataracts and provide new ideas for potential targets for drug discovery and development.